This project is to determine mechanisms by which the secondary intracellular metabolite, cyclic diguanylate (c-di-GMP), stimulates the formation of biofilms in the opportunistic pathogen Pseudomonas aeruginosa. Biofilms, defined as surface-associated multicellular communities encased in a self-produced extrapolysaccharide (EPS) matrix, are responsible for chronic P. aerugionsa infections in humans with underlying predispositions such as cystic fibrosis. Biofilm infections are problematic because they are resistant to antibiotic treatment and tend to escape immune surveillance. P. aeruginosa cells with high c-di-GMP form thick biofilms and distinctive wrinkled colonies and have decreased motility. The major biofilm-related effect of c-di-GMP in P. aeruginosa is to stimulate Psl and Pel EPS production. This is partly due to effects of c-di-GMP on pel and psl gene expression. C-di-GMP also stimulates the activities of the EPS biosynthetic enzymes. This effect is likely due to the compartmentalized production of c-di-GMP at specific subcellular sites by specific diguanylate cyclases. Although the general parameters of c-di-GMP activity have been established, the mechanisms of c-di-GMP action are just beginning to be explored. Proposed experiments will test the model that the transcription factor FleQ responds to c-di-GMP in concert with FleN to regulate transcription. An in vitro system with purified proteins and pel promoter DNA will be the starting point for experiments to explore mechanism (aim 1). Other experiments will characterize the catalytic properties of WspR, the most active diguanylate cyclase from P. aeruginosa. WspR is a hybrid response regulator-diguanylate cyclase that synthesizes c-di-GMP when activated by phosphorylation. wspR mutants are defective in EPS synthesis. Constitutively active mutant forms of WspR will be particularly targeted for characterization (aim 2). Fluorescent protein-tagged WspR forms dynamic clusters in the cytoplasm of cells when it is phosphorylated and therefore active. This indicates that WspR-P produces c-di-GMP at discreet subcellular locations and implies that cells have specific WspR-associated targets of c-di-GMP action. The subcellular locations of known c-di-GMP receptor proteins and their possible interactions with WspR will be assessed. Experiments to identify cytoskeleton proteins that may guide the distribution of WspR-P will also carried out. In addition, fluorescence microscopy will be used to establish structural features of WspR itself that are important for the subcellular localization and in vivo function of this diguanylate cyclase (aim 3). In past work it has been shown that P. aeruginosa cells with negligible intracellular c-di-GMP are unable to initiate biofilm formation. The work proposed here to elucidate mechanisms of c-di-GMP-mediated effects on cellular physiology could be important for the development of c-di-GMP based therapeutics to prevent biofilm infections. PUBLIC HEALTH RELEVANCE: Multicellular communities of bacteria called biofilms are responsible for chronic infections in humans with underlying predispositions such as cystic fibrosis. Biofilm infections are difficult to treat with antibiotics and tend to escape the human immune system. This research will explore how a small molecule called cyclic-di-GMP might be a target for preventing or treating biofilm infections.